The overall goal of our research is to understand the origin, development and regulation of distinctly functional human macrophages. The focus of this proposal is to use human pluripotent stem cells (hPSCs) to identify the essential genes for tissue resident macrophage development. Currently, the ontogenic origins for tissue-resident macrophage specification during embryonic development remains a poorly understood process. Our proposed studies build on our recent advances in the directed differentiation of extra-embryonic-like and intra-embryonic- like definitive hematopoietic progenitors from hPSCs, having identified a critical, stage-specific role for WNT signaling to regulate the specification of either population. With this methodology, we have now identified that both programs harbor robust, but phenotypically distinct, macrophage potential. As both ontogeny and local microenvironment are critical determinants to cell fate and function, this represents a powerful platform to study the role both factors, in an isogenic setting, impact human macrophage development. We hypothesize that ontogenically distinct hematopoietic progenitors are functionally imprinted, at the level of chromatin accessibility, dictating their ability to develop into anti-inflammatory macrophages, which is then further modified by local microenvironment. We will test this hypothesis across 2 Specific Aims. In Aim 1, we will assess the role of critical developmental regulators, such as MYB or GATA2, play on the specification of each hematopoietic program, with respect to macrophage phenotype, and chromatin accessibility. The objective of these studies is to establish the molecular regulation of ontogenically distinct macrophage progenitors. In Aim 2, we will define the contribution of fetal-like microenvironments on the development of tissue-resident macrophages, at a functional and genetic level. The objective of these studies is to determine whether tissue environment, or ontogenic origin, is the primary determinant to macrophage inflammatory potential. The successful completion of these studies will provide us with a more comprehensive understanding of macrophage development. This is of fundamental importance to basic biology, and the insights generated from these studies will have clinical implications, such as the in vitro generation of macrophages for a wide array of regenerative medicine applications. Our unique cellular and molecular tools, combined with our expertise in hPSC-derived hematopoietic development, immunology and bioinformatics puts us in an ideal position to make a significant impact in this field.